The present invention relates to the use of non-primate mammalian cell lines having substantially no endogenous retroviral sequences as producer and packaging lines for preparation of human serum-resistant retroviral vector particles. These cell lines have improved safety for use in gene therapy applications and can produce high titers of RVP. In a preferred embodiment, the cell line used in the present invention is the xcex1-galactosyl (xcex1Gal)-positive, ferret brain cell line designated as Mpf or a cell line having those identifying characteristics of the Mpf cell line suitable for the practice of the invention.
Retroviral vector particles (RVP) are functional retrovirus particles engineered to carry heterologous genes. Since RVP are capable of integrating into host mammalian cells as proviral DNA and expressing the heterologous (or foreign) gene, they have found use as therapeutic delivery agents in gene therapy. RVP have several advantages for gene therapy including the ability to efficiently transduce target cells, including human target cells, and integrate into the genomes of those cells at a frequency higher than most other systems. Other advantages include stable expression of the transduced genes, the capacity to transfer large genes, the lack of cellular cytotoxicity and the capacity to transduce mammalian cells from a wide variety of species and tissues.
To produce RVP, a gene of interest is inserted into a retrovirus vector. This vector is introduced into a retroviral packaging cell line to generate a retroviral producer cell line which in turn yields the RVP. Packaging cell lines express retroviral env and gag/pol genes, whereas producer cell lines are essentially packaging cell lines which additionally contain a retrovirus vector. Though not preferred, cell lines which contain only the retroviral vector are useful in some instances since they can be infected with a helper retrovirus. The RVP from such producer lines are, however contaminated with replication competent retrovirus (RCR). Collectively, the retrovirus vector, packaging and producer cell lines and RVP are referred to as a retroviral transduction system.
RVP produced in murine and many other species are not suitable for in vivo gene therapy or ex vivo gene therapy done in the presence of human body fluids (e.g., human serum) because these RVP are lysed in the presence of human serum. The cause of RVP virolysis has been actively investigated and appears to be mediated by the human complement system. For a review of the human complement system and various inhibitors thereof see U.S. Pat. Nos. 5,562,904 and 5,643,770. Some work has demonstrated that the presence of specific viral envelope proteins in the RVP is largely responsible for virolysis, while other work has suggested that the presence of the xcex1Gal sugar moieties are the sole or major factor responsible. Still other studies point to multiple factors including unknown packaging cell-specific factors. Based on the demonstration that anti-xcex1Gal antibodies in human serum inactivate retroviruses produced from animal cells via a complement-mediated pathway and related experiments (Rother et al. (1995) J. Exp. Med. 182:1345-1355; Takeuchi et al. (1996) Nature 379:85-88), Takeuchi and co-workers have suggested that viral vectors, including retroviral vectors, for human in vivo gene therapy should be prepared from xcex1Gal-negative cells (Takeuchi et al. (1997) J. Virol. 71:6174-6178).
Some human cell lines bearing retroviral envelope proteins have been identified which generate serum-resistant RVP; however, this is not a global feature of all human cell lines. In fact, no human cell line has been identified which universally generates serum resistant RVP for the different types of envelope proteins incorporated in the RVP. For example, the human cell line HT1080-Ampho is only 26% resistant to human serum.
While RVP can be made resistant to inactivation by human serum when produced in certain human and Old World primate cell lines, use of these cell lines raises safety related concerns. Human and primate cell lines contain large amounts of endogenous retroviral sequence. In some instances, endogenous retroviral sequences are expressed at the RNA level and in other instances cells shed viral particles or infectious endogenous viruses. It has been reported that up to 1% of RVP can carry inadvertently packaged endogenous retroviral sequences of producer cell origin which can then be effectively transduced into target cells. Replication-defective RVP, bearing only gag-pol or env sequences, have been identified in RVP supernatents and these have been associated with the formation of recombinant, infectious RVP in conjunction with endogenous sequences. Moreover, the demonstration that co-packaged RNA from different viral or endogenous sequences can interact and generate hybrid viruses makes the identification of non-primate cell lines capable of generating high titer, human serum-resistant RVP of significant importance. Use of non-primate cell lines which harbor fewer-endogenous retroviral sequences than conventional packaging cell lines would be an important safety advance in packaging and producer cell line development.
A variety of diseases may be treated by therapeutic approaches that involve stably introducing a gene into a cell such that the gene may be transcribed and the gene product may be produced in the cell. Diseases amenable to treatment by this approach include inherited diseases, particularly those diseases that are caused by a single gene defect. Many other types of diseases, including acquired diseases, may also be amenable to gene therapy. Examples of such acquired diseases include many forms of cancer, lung disease, liver disease, blood cell disorders and vascular disorders. See Anderson (1992) Science 256:808-813; Miller (1992) Nature 357:455-460; and Mulligan (1993) Science 260:926-932.
Delivery of the gene or genetic material into the cell is the first step in gene therapy treatment of disease. A variety of methods have been used experimentally to deliver genetic material into cells. Most research has focused on the use of retroviral and adenoviral vectors for gene delivery. Crystal (1995) Science 270:404-410. As discussed above, RVP are particularly attractive because they have the ability to stably integrate transferred gene sequences into the chromosomal DNA of the target cell and are very efficient in stably transducing a high percentage of target cells.
Most gene therapy protocols involve treating target cells from the patient ex vivo and then reintroducing the cells into the patient. Patients suffering from several inherited diseases that are each caused by a single gene defect have already received gene therapy treatments. Such treatments generally involve the transduction of the patient""s cells in vitro using RVP designed to direct the expression of therapeutic molecules, followed by reintroduction of the transduced cells into the patient.
For many diseases, however, it will be desired or necessary to introduce the gene into the target cell in situ, because the target cells cannot be removed from and returned to the body. For example, treatment of ischemic tissue can be done in situ via catheter delivery or direct injection of the gene therapy vector. In other cases, cells that are removed from the patient must be maintained in the presence of body fluids until returned to the body. Stem cells, particularly hematopoietic stem cells, are an especially important type of target cell for gene therapy of inheritable and acquired blood disorders. Such cells are intrinsically unstable in vitro, and tend to differentiate into cells that are less attractive targets for gene therapy, especially when they have been washed free of the fluids that surround them in vivo and transferred into body fluid-free tissue culture media or the like. For example, to transduce stem cells as quickly as possible, ex vivo treatment of such cells with RVP is best carried out in the cells natural milieu, i.e., in cells that have not been washed or otherwise removed from the body fluids in which they are obtained, e.g., hematopoietic stem cells in bone marrow aspirates.
The present invention is directed to a method for preparing a stable, retroviral packaging cell line for generation of human serum-resistant retroviral vector particles (RVP) which comprises introducing one or more packaging vectors into a non-primate mammalian cell line and recovering that cell line. The cell line of the invention exhibits substantially no hybridization to a Moloney-MLV retrovirus probe under stringent washing conditions and is capable of producing human-serum-resistant RVP. The packaging vectors of the invention, either singly or collectively, express a cellular targeting protein and retroviral gag and pol genes in amounts sufficient to package the RVP. In one embodiment the cell line is the Mpf cell line designated by ATCC accession number 1656CRL. The cellular targeting protein expressed by the packaging vectors of the invention is an amphotropic retroviral env protein, a xenotropic retroviral env protein, a polytropic retroviral env protein, a JSRV env protein, vesicular stomatitis virus G protein or transferrin. The invention also includes the packaging cell lines produced by this method.
The invention further provides a method for preparing stable, retroviral producer cells capable of producing human serum-resistant RVP which comprises introducing a retrovirus vector into the packaging cell line of the invention and recovering the producer cells. The retrovirus vector introduced into the packaging cell line is capable of being packaged into an RVP and comprises a heterologous gene capable of expression in a human. The invention also includes the producer cells produced by this method and RVP prepared from these producer cells.
Another aspect of the invention relates to a method of preparing human serum-resistant RVP which comprises introducing a retrovirus vector into the packaging cell line of the invention, culturing the cell line for a time and under conditions sufficient to produce the RVP and recovering the RVP. This method produces RVP having a supernatant titer on mink cell line Mv-1-Lu of at least about 104 to about 108 colony forming units per milliliter. The invention also includes the RVP produced by this method.
In another method, human serum-resistant RVP are prepared by a method which comprises culturing the producer cells for a time and under conditions sufficient to produce RVP and recovering the RVP. The invention also includes the RVP produced by this method.
Another aspect of the invention relates to a method for transducing a cell with a retroviral vector in the presence of a body fluid which comprises administering the RVP of the invention to the cell to be transduced. The RVP can be administered to the cell er vivo or in vivo.
Yet another aspect of the invention provides a method of gene therapy which comprises delivering a therapeutic molecule encoded on a retrovirus vector to a human cell via an RVP of the invention. Preferably, the therapeutic molecule is a hormone, a growth factor, an enzyme, a lymphokine, a cytokine, a receptor, an angiogenic factor, or an anti-angiogenesis factor. The RVP can be administered to the human cell ex vivo or in vivo.
Still another aspect of the invention is directed to a method for transferring a heterologous gene into a human cell which comprises contacting the human cell with producer cells of the invention under conditions such that the producer cells release RVP containing a retrovirus vector encoding the heterologous gene and thereby introduce that gene into the human cell. The producer cells can be implanted in a human, including in the human brain.
The invention also provides pharmaceutical compositions comprising the RVP of the invention with a pharmaceutically acceptable carrier.